Project Abstract In North America, there were an estimated 267,000 persons living with HIV infection (PLH) among 2 million persons who inject drugs in 2012. Opioids represent the dominant class of injected drug, and in 2013 517,000 adults reported heroin use within the past year, representing an approximately 150% increase compared to 2007. Medication assisted treatments (MAT) in the form of opioid agonist treatment with methadone or buprenorphine, or opioid antagonist treatment in the form of extended-release naltrexone (XR-NTX), together with other harm reduction services have greatly reduced HIV incidence by reducing opioid relapse, and contribute to significant public health improvement. The potential biologic effects of MAT agents on immune responses and chronic inflammation, particularly relevant in PLH with OUD, remain incompletely studied despite a substantial body of evidence for opioid-induced immunosuppression. More importantly, little work if any has evaluated the impact of MAT on HIV latency. We hypothesize that MAT in the form of opioid agonists, including methadone and buprenorphine; reactivate HIV-1 expression, while the opioid antagonist treatment of XR-NTX does not. We further hypothesize that the activation of opioid receptor signaling enhances HIV-1 reactivation through changing HIV-1 proviral and host genomic landscape. Using methods familiar to our research groups, we will carry out prospective, longitudinal studies of PLH with OUD starting MAT, recruited from the largest drug treatment centers in New Haven, Connecticut. We will obtain samples of blood at before MAT (day 0), and months 1 and 3 after the start of 3 three different FDA approved forms of MAT. Freshly collected samples of whole blood will be processed to assess HIV-1 RNA analysis, HIV-1 DNA analysis, HIV-1 HI-C and 4C-seq analyses. The R61 phase will be used to develop methods to examine HIV-1 expression, proviral landscape and genomic architecture in response to different forms of MAT among a sample of PLH with OUD. In Aim 1, we will examine HIV-1 expression level through quantitative and phylogenetic sequence analysis. In Aim 2, we will examine HIV-1 proviral landscape using limiting dilution sequencing. In Aim 3, we will examine the host chromosome architecture using Hi-C and HIV-1 4Cseq. If milestones are achieved at the conclusion of the R61 phase, then the R33 phase will be used to examine HIV-1 viral expression, proviral landscape and human genomic architecture among a larger cohort of PLH with OUD before and during treatment with the three forms of MAT using methods developed in the R61 phase. Taken together, we expect these studies to provide new information on the effects of MAT on parameters of HIV latency that should help in the development of guidelines for the selection of specific MAT agents among PLH with OUD. Our combination of expertise in treatment of OUD, HIV-1 latency and human genomics make it highly likely that the proposed studies will achieve a key goal of this RFA: ?to yield foundational knowledge that may inform the development of a future HIV cure for patients with SUDs.?